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Ligate DNA
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==Ligation Protocol== | ==Ligation Protocol== | ||
| + | Ligations attach two pieces of DNA at the cut site of the previous digestion, utilizing overlapping DNA sequences. | ||
| - | 1. Enter the names of the plasmids from which the vector and insert is obtained in the Excel worksheet. Also enter the lengths of the vector and insert fragments | + | |
| + | |||
| + | (For our lab) | ||
| + | |||
| + | |||
| + | 1. Enter the names of the plasmids from which the vector and insert is obtained in the Excel worksheet (created with calculations listed below). Also enter the lengths of the vector and insert fragments. | ||
2. The spreadsheet fills out the other columns to do a ligation with 100 ng of the vector fragment and a 1:3 molar ratio of vector to insert DNA. | 2. The spreadsheet fills out the other columns to do a ligation with 100 ng of the vector fragment and a 1:3 molar ratio of vector to insert DNA. | ||
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3. All ligations are typically done at RT for 2 hours, however difficult ligations or ligations with low concentration of vector or inserts (<10 ng/µl) can be done for 16 hours at 16C. | 3. All ligations are typically done at RT for 2 hours, however difficult ligations or ligations with low concentration of vector or inserts (<10 ng/µl) can be done for 16 hours at 16C. | ||
| - | 4. | + | 4. Keep the buffer on ice at all times when out of -20C. Keep the enzyme in coolers at all times when out of the -20C. |
| + | |||
| + | |||
| + | '''Specific Ligation Calculations''' | ||
| + | |||
| + | 1. Make a chart of the ODs and concentrations of your backbone and your insert. Add a column for Molecular weight. | ||
| + | |||
| + | Concentration = ng/ul | ||
| + | |||
| + | 2. Calculate molecular weights by using this formula: | ||
| + | |||
| + | (# nucleotides)/2628 x (1.75*10^6) = g/mole (or ng/nmole) | ||
| + | |||
| + | 3. Calculate nmole/ul (concentration/MW) for each | ||
| + | |||
| + | 4. Calculate the number of moles in 100 ng of backbone. | ||
| + | |||
| + | 5. Multiply that number by 3,4, or 5 (I don't know what molar ratio iGEM typically uses....I use either 3 or 5 molar ratio. The ratio is this: 5:1 insert:backbone (in moles). | ||
| + | |||
| + | 6. Now you have the number of moles you need for the inserts. Convert that number to the number of ul you need. Also figure out number of ul of backbone. | ||
| + | 7. Now you know the relative volumes for backbone and insert. From that, you can figure out how large your ligation reaction needs to be. You will have a 10x buffer and the T4 ligase in addition to your insert and backbone and you will also bring the volume up to the proper amount (if necessary) with sterile water. | ||
| - | + | 8. Mix gently and leave at room temp (labeled well!) for 2 hours. | |
Latest revision as of 00:59, 30 October 2008
PRINCETON IGEM 2008
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Ligation Protocol
Ligations attach two pieces of DNA at the cut site of the previous digestion, utilizing overlapping DNA sequences.
(For our lab)
1. Enter the names of the plasmids from which the vector and insert is obtained in the Excel worksheet (created with calculations listed below). Also enter the lengths of the vector and insert fragments.
2. The spreadsheet fills out the other columns to do a ligation with 100 ng of the vector fragment and a 1:3 molar ratio of vector to insert DNA.
3. All ligations are typically done at RT for 2 hours, however difficult ligations or ligations with low concentration of vector or inserts (<10 ng/µl) can be done for 16 hours at 16C.
4. Keep the buffer on ice at all times when out of -20C. Keep the enzyme in coolers at all times when out of the -20C.
Specific Ligation Calculations
1. Make a chart of the ODs and concentrations of your backbone and your insert. Add a column for Molecular weight.
Concentration = ng/ul
2. Calculate molecular weights by using this formula:
(# nucleotides)/2628 x (1.75*10^6) = g/mole (or ng/nmole)
3. Calculate nmole/ul (concentration/MW) for each
4. Calculate the number of moles in 100 ng of backbone.
5. Multiply that number by 3,4, or 5 (I don't know what molar ratio iGEM typically uses....I use either 3 or 5 molar ratio. The ratio is this: 5:1 insert:backbone (in moles).
6. Now you have the number of moles you need for the inserts. Convert that number to the number of ul you need. Also figure out number of ul of backbone.
7. Now you know the relative volumes for backbone and insert. From that, you can figure out how large your ligation reaction needs to be. You will have a 10x buffer and the T4 ligase in addition to your insert and backbone and you will also bring the volume up to the proper amount (if necessary) with sterile water.
8. Mix gently and leave at room temp (labeled well!) for 2 hours.
